CN110716049A - Gastric cancer serum detection marker and detection method and application thereof - Google Patents

Abstract

The invention discloses a gastric cancer serum detection marker, which is a polypeptide molecule with a sequence shown as SEQ ID NO. 1. The invention also discloses a detection method of the marker, a kit containing the marker and application of the marker. The polypeptide molecular fragment which shows high specificity expression in the serum of an early gastric cancer patient is obtained by screening, can be used as a serum detection marker for auxiliary diagnosis and detection of early gastric cancer, and thus, the possibility is provided for early discovery, diagnosis and treatment of gastric cancer.

Description

Gastric cancer serum detection marker and detection method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to detection of gastric cancer, and more particularly relates to a serum polypeptide molecule related to gastric cancer and a detection method and application thereof.

Background

The incidence and mortality of gastric cancer are the first of malignant tumors in China, and the current situation of the prevention and treatment of gastric cancer in China is 'two-high three-low', namely high incidence, high mortality, low early diagnosis rate, low radical operation rate and low survival rate in 5 years. Among the cases of gastric cancer, early gastric cancer accounts for only 2% -10%, and China has a considerable gap in the prevention and treatment of gastric cancer compared with Japan and Korea, which have advanced levels of international prevention and treatment of gastric cancer. It is statistically estimated that more than 70% of the detected stomach cancers in the medical institutions belonging to the japan early stomach cancer diagnosis association in 2004 are early stomach cancers, and such a high early stomach cancer detection rate is attributed to the screening of asymptomatic japan population for stomach cancer. The early gastric cancer early warning system is urgently needed to be effectively screened, the early gastric cancer early warning system is established, the early gastric cancer diagnosis rate is improved, and the screening and establishment of early gastric cancer markers play an important role.

The serum detection is a minimally invasive detection mode, and the occurrence and development conditions of tumors can be judged by detecting the markers in the serum, particularly protein markers in the serum, so that a reliable auxiliary means is provided for diagnosis and treatment, and particularly, the early diagnosis and treatment of the control have important functions. The currently accepted tumor markers in serum, such as CEA, CA125, AFP, etc., play an important role in the screening of tumors, but these several markers lack sensitivity and specificity in the screening of gastric cancer.

Serum proteomics is an important branch of clinical proteomics, and is a supplement to other functional genomics methods, and has been studied in a large amount in disease diagnosis, but research on early gastric cancer detection markers is just started.

Disclosure of Invention

The invention aims to solve the technical problem of providing a gastric cancer serum detection marker which presents high specificity expression in serum of patients with early gastric cancer and can assist in detecting early gastric cancer.

In order to solve the technical problem, the gastric cancer serum detection marker is a polypeptide molecule, and the amino acid sequence of the polypeptide molecule is shown as SEQ ID NO. 1.

The second technical problem to be solved by the present invention is to provide a method for detecting the gastric cancer serum detection marker in serum. The method adopts an indirect competitive ELISA method, and specifically comprises the following steps:

1) synthesizing polypeptide according to the sequence shown in SEQ ID N0:1, and coating the polypeptide on an enzyme label plate;

2) adding a sealing liquid into each hole of the ELISA plate for sealing;

3) adding the diluted serum sample into the closed reaction hole; adding a positive control and a negative control into the positive control well and the negative control well, respectively; adding the diluted enzyme-labeled polypeptide antibody into a corresponding hole on an enzyme-labeled plate;

4) preparing a TMB color developing agent, and adding the TMB color developing agent into each reaction hole of the ELISA plate for reaction;

5) adding a stop solution into each reaction hole of the ELISA plate, and stopping the reaction;

6) and (3) placing the ELISA plate in an ELISA reader for reading, evaluating a detection result according to the reading value, and judging whether the sample contains a polypeptide molecular marker with a sequence shown by SEQ ID N0: 1.

The coating method in the step 1) is preferably: diluting the polypeptide to 1 mu g/ml by using a coating buffer solution, adding 50 mu l of the polypeptide into an enzyme label plate, standing overnight at 4 ℃, discarding the solution in a hole the next day, washing 1 time by using a 1xTBST washing buffer solution at 180 mu l per hole, and patting to dry; the coating buffer preferably comprises: NaHCO 2₃2.93g，Na₂CO₃1.59g, deionized water was added to 1L.

The enzyme-labeled polypeptide antibody in the step 3) is preferably a polypeptide antibody labeled with HRP.

The above step 4), the TMB color developer is formed by mixing the color developing solution a and the color developing solution B immediately before use, and the volume ratio of the color developing solution a to the color developing solution B is preferably 1: 1. The color developing solution A preferably comprises the following components: 0.2M Na₂HPO₄25.7ml, 0.1M citric acid 24.3ml, H₂O₂0.1％、H₂O50 ml; the color developing solution B preferably contains: 3.9g of TMB, 10.52g of citric acid, 1.86g of EDTA, 2000ml of glycerol, 300ml of DMSO, and H₂O to 10000 ml.

The determination method in the step 6) is preferably: and setting the threshold value of the detection result as 1/3 of the detection value of the negative control hole, and if the detection value of the sample hole is lower than the threshold value, judging that the detected sample contains the polypeptide molecular marker of the sequence shown in SEQ ID N0: 1.

The invention also provides a kit containing the gastric cancer serum detection marker, and the kit can be used for detecting gastric cancer serum by an ELISA method.

Further, the kit of the present invention may further comprise: sealing liquid, sample diluent, developing liquid A, developing liquid B, stop solution, enzyme-labeled antibody, washing liquid, developer, positive control, negative control and enzyme-labeled plate.

The sample diluent is preferably a 0.5-5.0% BSA solution.

The positive control is preferably a synthetic polypeptide diluted 1: 500. The negative control is preferably 1% BSA.

The fourth technical problem to be solved by the present invention is to provide the use of the above-mentioned gastric cancer serum detection marker. The gastric cancer serum detection marker can be applied to serum-assisted detection and diagnosis of early gastric cancer.

The polypeptide molecular fragment which shows high specificity expression in the serum of a patient with early gastric cancer is obtained by screening, and can be used as a serum detection marker for auxiliary diagnosis and detection of early gastric cancer, so that the possibility is provided for early discovery, early diagnosis and early treatment of gastric cancer.

Drawings

FIG. 1 is an LC-MS/MS spectrum of the polypeptide fragment shown in SEQ ID NO. 1 in serum samples of different groups.

FIG. 2 is a Wien diagram of the distribution of polypeptides detected in different samples.

FIG. 3 is a schematic diagram of the ELISA kit used in example 2 of the present invention for detecting the polypeptide fragment represented by SEQ ID NO. 1.

FIG. 4 is a schematic diagram of the structure of a 96-well microplate in the kit of FIG. 3.

FIG. 5 is a schematic diagram illustrating the principle of the indirect competition ELISA method according to example 2 of the present invention.

The reference numerals are explained below:

1: sealing liquid

2: sample diluent

3: color developing solution A

4: color developing solution B

5: stopping liquid

6: HRP-labeled antibody

7: washing lotion

8: TMB color-developing agent

9: positive control

10: negative control

11: enzyme label plate

111: reaction well

112: disassembling line

13: sample polypeptides

14: polypeptide antibody

15: polypeptide coated on enzyme label plate

Detailed Description

In order to more specifically understand the technical content, characteristics and effects of the present invention, the technical solution of the present invention will be further described in detail with reference to the accompanying drawings and specific embodiments. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the examples, generally following conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations.

Example 1 gastric cancer serum marker screening

1. Sample collection

Collecting 10 cases of gastric cancer patients and normal control people, collecting whole blood 5ml, standing at room temperature for 30min, centrifuging at room temperature for 5min (3000g), collecting upper layer serum 200 μ l/tube, storing at-80 deg.C, and avoiding repeated freeze thawing.

2. Serum sample pretreatment

Adding 25% acetonitrile with 3 times volume (600 μ l) into 200 μ l of serum, and standing at 4 deg.C for 40 min; adding 2.5 times volume of 100% acetonitrile, precipitating at-20 deg.C for 2 hr, centrifuging at 12000rpm at 4 deg.C for 30min, and collecting supernatant and precipitate respectively. The supernatant was centrifuged, concentrated and dried to obtain a precipitate, which was washed with 300. mu.l of 50mM NH₄HCO₃And (4) re-dissolving.

3. Ultrafiltration

Centrifuging at 12000rpm at 4 deg.C for 10min, collecting supernatant, adding into 10kD ultrafiltration tube, and collecting filtrate.

4. Reductive alkylation, desalting and drying.

5. Analysis on computer

The dried sample was added with 10. mu.l of loading buffer (99.9% water, 0.1% formic acid), shaken for 10min to redissolve, centrifuged, and 5. mu.l of sample was added to a sample bottle for loading analysis. Adopting nano-liter flow rate HPLC liquid phase system EASY-nLC 1000 for each sample

(Thermo Scientific, USA) and then through a Trap column and then a C18 analytical column at a flow rate of 300 nl/min. Desalting by HPLC and separation were analyzed by an Orbitrap Elite mass spectrometer, and the mass spectrum results are shown in FIG. 1.

6. Polypeptide screening

The results obtained from the mass spectrometry were subjected to a search analysis using the protocol discovery 1.4 library (Thermo Scientific, USA) with the parameters shown in Table 1 and identified as a list of polypeptides as shown in Table 2.

TABLE 1 Proteome Discoverer1.4 library analysis parameters

TABLE 2 polypeptide List

After the uniform combination, 147 uniquepeptides were obtained from the normal group, and 119 uniquepeptides were obtained from the gastric cancer case group. The distribution of unique peptides is shown in FIG. 2.

The obtained polypeptides were subjected to statistical analysis to find out different polypeptides, wherein the different polypeptides ranked at position 1 are shown in Table 3.

TABLE 3 significantly different Polypeptides

The polypeptide molecules in Table 3 (amino acid sequence shown in SEQ ID NO: 1) have significant differences. The expression level of the polypeptide shown by the sequence in peripheral serum of a case group and a normal group is detected by LC-MS/MS, and the signal intensity of the polypeptide in the case group of the gastric cancer is far higher than that in the normal group (N18), as shown in Table 4.

TABLE 4

The polypeptide with the significant difference of the sequence shown in the SEQ ID N0:1 is a fragment of human Inter-Alpha Trypsin Inhibitor Heavy Chain H4(Inter-Alpha-Trypsin Inhibitor Heavy Chain H4, ITIH4) protein, and the precise molecular weight of the polypeptide is 2581.3 daltons. By analyzing the frequency of the polypeptide in the sample and the detected signal intensity, the polypeptide shows specific high expression in the peripheral serum of the early gastric cancer patient: the serum detection rate of the healthy group is 10 percent (1/10), the serum detection rate of the early gastric cancer group is 80 percent (8/10), the specificity is obvious (p is less than 0.001), and the serum can be used as a potential serum marker for the auxiliary diagnosis of early gastric cancer.

Example 2 detection of gastric cancer-associated polypeptide molecular markers in serum by Indirect competitive ELISA

The principle of indirect competitive ELISA (enzyme linked immunosorbent assay) of this embodiment is shown in fig. 5, wherein the polypeptides in the sample compete with the polypeptides coated on the ELISA plate together for reaction with the antibodies labeled with HRP (horse radish peroxidase), i.e. only the remaining antibodies bound with the polypeptides in the test sample can bind with the polypeptides coated on the ELISA plate, the antibodies not reacted with the polypeptides coated on the ELISA plate and the antibodies bound with the polypeptides in the test sample are washed away in the washing process, only the antibodies bound with the polypeptides coated on the ELISA plate will react with the TMB color developing agent to generate a test signal, and the intensity of the test signal is inversely proportional to the content of the polypeptides in the sample.

The specific operation steps are as follows:

1. 96-hole plate for coating

Preparing a coating buffer solution: NaHCO 2₃2.93g，Na₂CO₃1.59g, deionized water was added to 1L and stored at 4 ℃.

The polypeptides synthesized according to the sequence shown in SEQ ID N0:1 were diluted to 1. mu.g/ml with coating buffer, 50. mu.l were added to a 96-well plate of ELSIA plate overnight at 4 ℃, the well solutions were discarded the next day, washed 1 time with 180. mu.l per well with 1 XTSST washing buffer and patted dry.

And (4) storing the coated 96-well plate at-20 ℃ for later use.

2. Prepared color developing liquid

Color developing solution A: 0.2M Na₂HPO₄25.7ml, 0.1M citric acid 24.3ml, H₂O₂0.1％，H₂O 50ml

Color developing solution B: 3.9g of TMB, 10.52g of citric acid, 1.86g of EDTA, 2000ml of glycerol and 300ml of DMSO. After the above components were dissolved (note that TMB was first dissolved in DMSO), H was added₂O to 10000 ml.

TMB color development liquid: the color developing liquid A and the color developing liquid B are mixed according to the volume ratio of 1:1 before use.

3. Sample detection

1) And taking out the 96-well enzyme label plate from the kit, splitting the next 8-well plate, adding 60 mu l of sealing solution into each well for sealing, incubating at 37 ℃ for 1 hour, and then removing the sealing solution.

2) Pretreatment of a serum sample: taking 50 mul serum, adding 50 mul diluent for dilution;

3) a50. mu.l sample of diluted serum was taken and added to the above-mentioned blocked reaction well. Positive control wells (plus positive control-synthetic polypeptide diluted 1: 500) and negative control wells (plus negative control-1% BSA) were also set. Incubate at 37 ℃ for 30min, pat dry, wash 2 times with 180. mu.l per well of 1 XTSST wash buffer, pat dry.

4) Adding an enzyme-labeled antibody: the diluted polypeptide antibody-HRP (diluted with a diluent at a ratio of 1: 3000) was added at 50. mu.l/well to the wells of an 8-well microplate, incubated at 37 ℃ for 30min, then discarded, washed 3 times with 180. mu.l/well of washing buffer, and blotted dry.

5) Adding a TMB color developing agent for color development: mixing 50 mul of color developing solution A and 50 mul of color developing solution B respectively to prepare TMB color developing agent, adding 100 mul of prepared TMB color developing agent into each reaction hole, reacting at 37 ℃ for 5min, and stopping reaction.

6) And (3) placing the 8-hole enzyme label plate in a preheated enzyme label instrument for reading, and evaluating a detection result according to the reading value. And setting the threshold value of the detection result as 1/3 of the detection value of the negative control hole, and if the detection value of the sample hole is lower than the threshold value, judging that the detected sample contains the sequence shown in SEQ ID N0: 1.

The results of the 6 serum samples are shown in Table 5, wherein the sample 6 with a value below the threshold was judged to contain the detected polypeptide sequence shown in SEQ ID N0: 1.

TABLE 5

Sample 1	Sample 2	Sample 3	Sample 4	Sample 5	Sample 6	Positive control well	Negative control well
								1.326	1.287	1.276	0.996	0.969	0.301	0.287	1.287

Sequence listing

<110> first-person hospital in Shanghai City

Shanghai Huaying biological medicine science and technology Limited

<120> gastric cancer serum detection marker and detection method and application thereof

<130>LHJ-NP-18-100023

<160>1

<170>SIPOSequenceListing 1.0

<210>1

<211>23

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>1

Asn Val His Ser Gly Ser Thr Phe Phe Lys Tyr Tyr Leu Gln Gly Ala

1 5 10 15

Lys Ile Pro Lys Pro Glu Ala

20

Claims (10)

1. The gastric cancer serum detection marker is characterized in that the marker is a polypeptide molecule, and the amino acid sequence of the polypeptide molecule is shown as SEQ ID NO. 1.

2. The method for detecting a marker according to claim 1, wherein an indirect competitive ELISA method is used, and the method comprises the steps of:

1) synthesizing polypeptide according to the sequence shown in SEQ ID N0:1, and coating the polypeptide on an enzyme label plate;

2) adding a sealing liquid into each hole of the ELISA plate for sealing;

3) adding the diluted serum sample into the closed reaction hole; adding a positive control and a negative control into the positive control well and the negative control well, respectively; adding the diluted enzyme-labeled polypeptide antibody into a corresponding hole on an enzyme-labeled plate;

4) preparing a TMB color developing agent, and adding the TMB color developing agent into each reaction hole of the ELISA plate for reaction;

5) adding a stop solution into each reaction hole of the ELISA plate, and stopping the reaction;

6) and (3) placing the ELISA plate in an ELISA reader for reading, evaluating a detection result according to the reading value, and judging whether the sample contains a polypeptide molecular marker with a sequence shown by SEQ ID N0: 1.

3. The method of claim 2, wherein step 1), the coating method is: diluting the polypeptide to 1 mu g/ml by using a coating buffer solution, adding 50 mu l of the polypeptide into an enzyme label plate, standing overnight at 4 ℃, discarding the solution in a hole the next day, washing 1 time by using a 1xTBST washing buffer solution at 180 mu l per hole, and patting to dry; the coating buffer comprises the following components: NaHCO 2₃2.93g，Na₂CO₃1.59g, deionized water was added to 1L.

4. The method according to claim 2, wherein in step 3), the enzyme-labeled polypeptide antibody is a polypeptide antibody labeled with HRP.

5. The method of claim 2The method is characterized in that, in step 4), the TMB color developing agent is formed by mixing a color developing solution A and a color developing solution B according to a volume ratio of 1:1, and the color developing solution A comprises the following components: 0.2M Na₂HPO₄25.7ml, 0.1M citric acid 24.3ml, H₂O₂0.1％、H₂O50 ml; the color developing solution B comprises the following components: 3.9g of TMB, 10.52g of citric acid, EDTA1.86g, 2000ml of glycerol, 300ml of DMSO, and H₂O to 10000 ml.

6. The method according to claim 2, wherein in step 6), the threshold of the detection result is set to 1/3 of the detection value of the negative control well, and if the detection value of the sample well is lower than the threshold, the polypeptide molecular marker having the sequence shown in SEQ ID No. 0:1 in the detected sample is determined.

7. Gastric cancer serum ELISA detection kit, characterized in that the kit comprises the gastric cancer serum detection marker of claim 1.

8. The kit of claim 7, further comprising: sealing liquid, sample diluent, developing liquid, stop solution, enzyme-labeled antibody, washing liquid, developer, positive control, negative control and enzyme-labeled plate.

9. The kit of claim 7, wherein the sample diluent is a 0.5-5.0% BSA solution; the color developing liquid comprises a color developing liquid A and a color developing liquid B, wherein the color developing liquid A comprises the following components: 0.2M Na₂HPO₄25.7ml, 0.1M citric acid 24.3ml, H₂O₂0.1％、H₂O50 ml, the color developing solution B comprises the following components: 3.9g of TMB, 10.52g of citric acid, 1.86g of EDTA, 2000ml of glycerol, 300ml of DMSO, and H₂O to 10000 ml; the color developing agent is formed by mixing a color developing solution A and a color developing solution B according to the volume ratio of 1:1 before use; the enzyme-labeled antibody is a polypeptide antibody marked with HRP; the positive control was a 1:500 dilution of the synthetic polypeptide and the negative control was 1% BSA.

10. The use of the serum detection marker for gastric cancer according to claim 1 for early-stage serum detection of gastric cancer.

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